Colorimetric determinations of glutamic transaminases in biological fluids are carried out by various methods in many laboratories. The value of such determinations as an aid in diagnosis of myocardial infarction and necrosis of hepatic cells is well established. Reitman and Frankel, Am. J. Clin. Pathol. 28, 56 (1957). The enzymes of primary significance are glutamic oxalacetic transaminase (GOT), which catalyzes the reaction L-Aspartate + .alpha.-Oxoglutarate .revreaction. Oxalacetate + L-Glutamate and the enzyme glutamic pyruvic transaminase (GPT) which catalyzes the reaction
L-Alanine + .alpha.-Oxoglutarate .revreaction. Pyruvate + L-Glutamate The methods are based on the enzyme-catalyzed transamination of a substrate containing .alpha.-oxoglutarate and an appropriate amino acid to produce glutamate and another acid, pyruvate in the case of GPT and oxalacetate in the case of GOT.
Some colorimetric methods are based on a color reaction involving the acid produced. More recently, a method has been described in which glutamate produced in the transamination reaction is dehydrogenated, using the enzyme glutamate dehydrogenase, (G1DH) in aqueousl ammonium sulfate and the co-enzyme oxidized nicotinamideadenine dinucleotide (NAD), to form .alpha.-oxoglutarate, ammonia and reduced nicotinamide-adenine dinucleotide (NADH), Glutamate +H.sub.2 O+NAD .sup.GlDH .alpha.-Oxoglutarate +NH.sub.3 +NADH. The NADH produced is reacted with a colorless tetrazolium dye, 2-p-iodophenyl-3-nitrophenyl-5-phenyltetrazolium chloride (INT) with the aid of an electron carrier, such as N-methyl phenazonium methosulfate (PMS) to form a colored dye (INT formazan), in a reaction catalyzed by the electron carrier, as follows: EQU NADA + INT .sup.PMS NAD + INT formazan
The intensity of the color formed in then measured to give a measurement of the transaminase activity, Lippi and Guidi, Clin. Chim. Acta, 28, 431 (1970).
The transaminase determination is usually performed on a biological fluid such as serum in the differential diagnoses of liver and heart disease. A predetermined volume of sample fluid is mixed with a predetermined volume of a substrate composition buffered to a pH of about 7.4 and containing .alpha.-oxoglutarate and either aspartic acid (for GOT) or alanine (for GPT). A color developer solution or solutions containing the GlDH, INT and NAD are also employed. The substrate, sample and color developer are mixed in a container such as a colorimeter color developer cuvette or test tube. The mixture is incubated, typically in a water bath or heating block at 37.degree. C. for a predetermined period of time, about 45-60 minutes. The incubation is terminated by addition of acid and the intensity of the resulting color is measured photometrically in a spectrophotometer or colorimeter at a wavelength from 490 to 550 nanometers, and compared with a control serum, calibration curve or the like.